Liver Cancer Open Access
Copyright ©2007 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jan 14, 2007; 13(2): 228-235
Published online Jan 14, 2007. doi: 10.3748/wjg.v13.i2.228
Interferon-α response in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment
Shi-He Guan, Petra Grünewald, Guido Gerken, Jörg F Schlaak, Department of Gastroenterology and Hepatology, University Hospital of Essen, Essen, Germany
Shi-He Guan, Mengji Lu, Michael Roggendorf, Institute of Virology, University Hospital of Essen, Essen, Germany
Shi-He Guan, Department of Laboratory Medicine, the first Affiliated Hospital of Anhui Medical University, China
Author contributions: All authors contributed equally to the work.
Supported by grants from the Deutsche Forschungsgemeinschaft (DFG SCHL 377/2-2, LU 669/2-1 and GRK 1045/1)
Correspondence to: Jörg F Schlaak, MD, Professor of Medicine, Department of Gastroenterology and Hepatology, University Hospital of Essen, Hufelandstr. 55, Essen 45122,Germany. joerg.schlaak@uni-essen.de
Telephone: +49-201-7232518 Fax: +49-201-7235749
Received: August 8, 2006
Revised: August 25, 2006
Accepted: September 20, 2006
Published online: January 14, 2007

Abstract

AIM: To characterize the IFN-response and its modul-ation by the antiviral compound lamivudine in HBV-transfected HepG2.2.15 cells.

METHODS: HepG2.2.15 and HepG2 cells were stimulated with various concentrations of IFN-α2a in the presence or absence of lamivudine. Then, total RNA was extracted and analysed by customised cDNA arrays and northern blot for interferon-inducible genes (ISGs). In addition, cellular proteins were extracted for EMSA and western blot. HBV replication was assessed by southern blot or ELISAs for HBsAg and HBeAg.

RESULTS: Two genes (MxA, Cig5) with completely abolished and 4 genes (IFITM1, -2, -3, and 6-16) with partially reduced IFN-responses were identified in HepG2.2.15 cells. In 2 genes (IFITM1, 6-16), the response to IFN-α could be restored by treatment with lamivudine. This effect could not be explained by a direct modulation of the Jak/Stat signalling pathway since EMSA and western blot experiments revealed no suppression of Stat1 activation and ISGF3 formation after stimulation with IFN-α in HepG2.2.15 compared to HepG2 cells.

CONCLUSION: These results are consistent with the assumption that chronic hepatitis B may specifically modulate the cellular response to IFN by a selective blockage of some ISGs. Antiviral treatment with lamivudine may partially restore ISG expression by reducing HBV gene expression and replication.

Key Words: Hepatitis B; IFN-α; Gene expression; Lamivudine



INTRODUCTION

Hepatitis B (HBV) is a hepatotropic DNA virus capable of causing both acute and chronic hepatitis in humans. It is estimated that over 350 million people are chronically infected with HBV worldwide. Currently approved therapeutic strategies for treatment of HBV include interferon-alpha (IFN-α), the nucleoside analogue lamivudine and the nucleotide analogue adefovir[1,2]. However, only a minority of patients treated with IFN-α has a long-term sustained response with ‘eradication’ of the virus. Patients with a high viral load, in particular, rarely respond to IFN therapy. Treatment with lamivudine, on the other hand, is complicated by a high rate of viral resistance and a high relapse rate after cessation of therapy, respectively[3]. Both the emergence of viral resistance and relapse after therapy are often associated with a hepatitis flare, which can sometimes be fatal. Thus, novel strategies are needed to improve treatment for this disease.

To develop new regimens it is necessary to gain further insights into the interactions between HBV and the main antiviral system of the host, the IFN-system. It has been shown that typeIand type II interferons are able to suppress HBV-replication in livers from HBV-transgenic mice[4-6]. This could also be demonstrated in vitro by using immortalized hepatocyte cell lines from these animals[7] and involves elimination of pregenomic RNA-containing capsids, inhibition of DNA replication and reduction of steady-state levels of HBV transcripts. The effector mechanisms that have been associated with IFN-induced suppression of HBV-replication include MxA[8] and proteasome mediated activities[9,10]. Additional data suggest a role for GTP-binding proteins, signalling and various other molecules in the control of HBV replication[11]. HBV can counteract these antiviral effector mechanisms by inhibiting proteasome activities in an HBX-dependent manner[12] and by suppressing MxA expression at the promoter level[13]. Furthermore, it has been shown that HBV replicated at higher levels in HBV-transgenic mice crossed with IRF-1 or PKR deficient mice while replication was unchanged in transgenic mice crossed with RNase L deficient mice[14].

Assuming that HBV may interfere with the expression of ISGs, one would predict that the ISG expression in cell lines with and without HBV may be different and this would be modulated by inhibition of HBV gene expression and replication. The present study was performed to test this hypothesis. Using customized cDNA arrays for ISGs, we could identify 2 ISGs (MxA and Cig5) that are completely abolished in HBV-transfected HepG2.2.15 cells and 4 genes (IFITM1, -2, -3 and 6-16) with partially reduced responses. This suppression could partially be restored in 2 genes (IFITM1, 6-16) by treatment with the nucleoside analogue lamivudine suggesting an additional therapeutic mechanism for this drug.

MATERIALS AND METHODS
Cell culture

HepG2.2.15 cells were kindly provided by G. Acs (Mount Sinai Medical Cancer, New York, NY) and maintained in Dulbecco’s Modified Eagle’s Medium, supplemented with 2 mmol/L L-glutamine 50 IU/mL of penicillin, 50 mg/L of streptomycin, 500 mg/L of G418, 5% (vol/vol) fetal bovine serum, at 37°C in humidified incubators at 5% CO2. The cells were seeded at a density of 8 × 105 cells and maintained in a confluent state for 2 to 3 d before being treated with antiviral compounds. At first, various concentrations from 0.04 μmol/L to 100 μmol/L of lamivudine were used to reach the suitable drug concentration, which profoundly suppressed HBV replication without cytotoxicity. At the same time, a time course of drug action also was evaluated. Over a period of 10 d lamivudine was added to the medium daily, then the cells were stimulated by addition of IFN-α for 6 h. Thereafter, the media were collected and DNA or RNA was extracted for further analysis.

Analysis of secreted HBV particles

Detection of HBsAg and HBeAg was carried out by using a commercially available kit (Dade Behring) according to the manufacturer’s instructions. Medium samples collected from HepG2.2.15 cells were centrifuged at 1200 rpm for 10 min to remove cellular debris, transferred to clean tubes and stored at -20°C until analysed. HBsAg and HBeAg amounts were evaluated from absorbance reading values (450 nm) compared to the constructed controls.

HBV DNA analysis

Extracellular virion HBV-DNA analysis: Medium of HepG2.2.15 cells was collected and centrifuged (10 min, 2000 × g), and polyethylene glycol (Mr, 8000) was added to the supernatant at a concentration of 10% (wt/vol) followed by overnight precipitation at 4°C. The virions were pelletted (30 min, 10 000 × g), and the pellet was resuspended in lysis buffer (10 mmol/L Tris-Cl, 5 mmol/L EDTA, 150 mmol/L NaCl, 1% SDS) at room temperature for 15 min. Proteinase K was added at a concentration of 500 μg/mL and the suspension incubated for 2 h at 56°C. The digest was extracted with phenol/chloroform, 1:1 (vol/vol) or chloroform, respectively, and the DNA was precipitated with 2.5 vol. of ethanol. The DNA pellet was dissolved in TE solution and then spotted onto Hybond-N+ membranes. Alternatively, the DNA was electrophoresed in 1.2% agarose gel followed by blotting onto Hybond-N+ membranes. The bolt was hybridized with a 32P-labeled HBV DNA probe (digested by NsiIfrom plasmids that contained the full length HBV genome sequence dimer and labelled with a RediprimeTM II Random prime labelling system), washed with 2 × SSC/0.1% SDS at room temperature for 20 min, twice, and 0.1 × SSC/0.1% SDS at 60°C for 45 min, and then autoradiographed. The intensity of the autoradiographic dots or bands was quantitated using the Cyclone Storage Phosphor System (Packard Instrument Company, Median, Conn.). All drug concentrations were tested in duplicate or triplicate, with antiviral effects being scored as the amount of HBV DNA present in the media relative to that in untreated controls.

Intracellular HBV replicative intermediates (RI) analysis: HepG2.2.15 cells were consecutively treated with various concentrations of lamivudine for 10 d. The cytoplasmic preparations containing HBV core particles were isolated from the treated cells. Cells were lysed with lysis buffer (50 mmol/L Tris-Cl, PH 7.4, 150 mmol/L NaCl, 5 mmol/L MgCl2, 0.5% NP-40) at room temperature for 5-10 min. The cytoplasmic fraction was separated from the nuclear fraction by centrifugation. Unprotected DNA was removed by adjusting cytoplasmic preparations so that they contained 10 mmol/L MgCl2 and 500 μg/mL of DNaseI(Roche, Germany) followed by a 1 h incubation at 37°C. To extract replicative intermediates (RI), EDTA, sodium dodecyl sulfate (SDS), NaCl and proteinase K (QIAGEN) were added separately and sequentially to final concentrations of 10 mmol/L EDTA, 1% SDS, 100 mmol/L NaCl and 500 mg/L of proteinase K. The sample was incubated for 1.5 h at 56°C and then subjected to sequential phenol and chloroform extraction and isopropanol precipitation. Precipitated nucleic acids were resuspended in a small volume of TE solution and digested with 100 mg/L of RNase (Roche, Germany) for 1 h at 37°C. Twenty micrograms of cytoplasmic preparations containing HBV replicative intermediates DNA (RI) were then analysed by electrophoresis in 1.2% agarose gels, followed by blotting onto Hybond-N+ membranes. The bolt was hybridized with a 32P-labeled HBV DNA probe (digested by NsiIfrom plasmids which contain full length HBV genome sequence dimer, and labelled with a RediprimeTM II Random prime labelling system), washed with 2 × SSC/0.1% SDS at room temperature for 20 min, twice, and 0.1 × SSC/0.1% SDS at 60°C for 45 min, and then autoradiographed as described above.

RNA extraction

Total RNA was isolated from cells using Trizol according to the manufacturer’s instructions. RNA quantity and quality was assessed by determination of the optical density at 260 and 280 nm using spectrophotometry and additional visualisation by agarose gel electrophoresis.

Gene expression profiling by customized cDNA macroarrays

Radiolabelled cDNA was generated from 20 μg total RNA by reverse transcription with SuperscriptII(Gibco, MD) in the presence of 32P-dCTP. Residual RNA was hydrolysed by alkaline treatment at 70°C for 20 min and the cDNA was purified using G-50 columns (Amersham Pharmacia, UK). Before hybridisation to the macroarrays the labelled cDNA was mixed with 50 μg COT-DNA (Gibco) and 10 μg Poly-A DNA (Sigma), denatured at 95°C for 5 min and hybridised for 1h to minimise non-specific binding. Preparation of the macroarrays (representing 150 known ISGs), hybridisation of the radioactive cDNAs and scanning and analysis of the macroarrays were carried out as described previously[15].

Northern blot analysis

5 μg of total RNA was electrophoresed through a 1.2% agarose gel containing formaldehyde and then transferred to Hybond-N+ membranes. The immobilized RNA was hybridized with a 32P-labeled DNA probe (IMAGE clones PCR products, purified with Gel Extract kit, QIAGEN).

Electrophoretic Mobility Shift Assay

At 80% to 90% confluence, cells were stimulated with IFN-α for 6 h. Preparations of nuclear extracts were performed according to the instruction of the manufacturer (PIERCE, NE-PERTM Nuclear Extraction Reagent). Nuclear extracts/DNA binding reactions were performed in 20 μL containing 15 μg nuclear extract protein and 4 μL Gel Shift Binding 5 × Buffer (20% glycerol, 5 mmol/L MgCl2, 2.5 mmol/L EDTA, 2.5 mmol/L DTT, 250 mmol/L Tris-Cl, PH 7.5, 0.25 mg/mL Poly (dI-dC)·Poly (dI-dC)). ISRE/GAS consensus oligonucleotides (5’-AAG TAC TTT CAG TTT CAT ATT ACT CTA-3’) from the promoter region of the IFN-α responsive genes were used. Mutant oligonucleotides (5’-AAG TAC TTT CAG TGG TCT ATT ACT CTA-3’) were used as control. The probes were end-labeled with γ-32P-ATP (U K, 3000 Ci/mol) at room temperature for 20 min. Complexes were separated from the probe in 4% naive poly-acrylamide gel in 0.5 × TBE buffer. The gels were subsequently dried and autoradiographed.

Western blot analysis

After interferon treatment, cells were washed once with ice-cold phosphate-buffered saline. Cells were lysed on ice for 30 min in 0.5 mL lysis buffer containing 50 mmol/L Tris, pH 8.0, 10% Glycerol, 0.5% NP40, 150 mmol/L NaCl, 1 mmol/L DTT, 1 mmol/L EDTA, 1 mmol/L Sodiumorthovanadate, 170 mg/L phenylmethylsulfonyl fluoride, 2 mg/L Aprotinin, 1 mg/L Leupeptin. Lysates were cleared by centrifugation in a microcentrifuge at high speed for 30 min at 4°C. Protein concentration of the supernatant was measured with Bradford reagent. Equal amounts (100 μg) of proteins were suspended in sodium-dodecyl sulphate (SDS)-sample buffer, boiled for 5 min and separated by electrophoresis (NuPAGE 4%-12% Bis-Tris Gel, Invitogen). The separated proteins were transferred to a polyvinylidene difluoride membrane (Hybond-PTM, Amersham Biosciences). After blocking for 1 h at room temperature in 10% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) or 1% BSA for antibodies specific for phosphorylated epitopes, membranes were incubated with anti-p38, anti-pp38 (Santa Cruz), anti-Stat1, anti-Stat1(pY701) and anti-ERK1, anti-ERK1/2(pT202/pY204) (BD Biosciences) overnight at 4°C, and thereafter with horseradish peroxidase-conjugated anti-rabbit or anti-Mouse IgG (1:5000) (Amersham Biosciences) for 1 h at room temperature. The proteins were detected with enhanced chemiluminescence reagent (ECL, Amersham).

Southern blot analysis

Twenty micrograms of cytoplasmic preparations containing HBV replicative intermediates (RI) DNA were analysed by Southern blotting as above.

RESULTS
Differential expression of ISGs in HepG2.2.15 and HepG2 upon stimulation with IFN-α

Type 1 IFNs are known to induce an intracellular antiviral state against many viruses. Therefore, we developed a customized cDNA array methodology to study the expression of IFN stimulated genes (ISGs). At present, this system permits the analysis of several hundred genes of interest. A substantial spectrum of known ISGs is analysed with this macroarray (Table 1). The sensitivity of this method has also been assessed previously[15]. Conventionally, in most micro- and macroarray systems a 2-fold change in the expression level is regarded as being significant.

Table 1 Complete list of genes investigated in this study.
Gene NameAcc. No.Gene NameAcc. No.Gene NameAcc. No.
101F6AA544950IFI 16M63838Mdm2Z12020
2-5 OASX02875IFI 41L22342MEN1U93237
2-5 OASD00068IFI 44D28915MetAA410591
5' nucleotidaseX55740IFI 6-16BC015603MigX72755
60S Ribosomal protein L11U43522IFI27X67325MIP-1b/CCL4NM_002984
72 kDa type IV collagenaseJ03210IFI4X79448MLK 2X90846
9-27J04164IFIT 1M24594MMP-1M13509
ADAM-10AF009615IFIT4U72882MxAM33882
ADAM-17U69611IFIT4AF083470MxAM33882
akt-1NM_005163IFITM2X57351MxBM30818
akt-2M77198IFITM3X57352MxBM30818
Alpha-1-antiproteinaseK01396IFN omega 1X58822NCAMM74387
Alpha-crystallinU05569IFN-AR1J03171NF-IL-6X52560
ATF-2X15875IFN-AR2L42243NFkBM58603
Auto Ag SS-A/RoNM_003141IFN-gM29383NKC-4M59807
badU66879IFN-GR1J03143n-mycY00664
BAK1X84213IFN-GR2U05875p19U40343
BAXU19599IFI 17J04164p48/ISGF3gM87503
BaxL22474IFP 35U72882p53M14694
bcl-2M14745IFP-53X62570p57Kip2U22398
BRCA1U14680IFRG28AJ251832p70 S6 kinaseM60724
BST2D28137ikBaM69043PAI-1M16006
BTG1X61123IL-1 αM28983PCBPM80563
CalcyclinJ02763IL-10M57627PDGF-alphaX06374
CalretiulinM84739IL-10 R αU00672PDK1Y15056
CASPAJ006470IL-10 R βZ17227PDK2NM_002611
Caspase 7U67319IL-12R βU64198Phosph. Scram. 1AF098642
Caspase 8X98172IL13RAU81379Phosph.glycerate kin.V00572
Caspase-1M87507IL13RA 2U70981Pi3-kinaseNM_006219
Caspase-9U60521IL-15U14407PIAS x-betaAF077954
Cat. o-methyltransferaseM58525IL-15RAU31628pig7AF010312
CBFANM_004349IL-18D49950pim-1M16750
CBPU85962IL-18 bprotAB019504PK RAF072860
CCR1L09230IL2U25676PKRU50648
CCR5U54994IL-2R αK03122plectin (PLEC1)U53204
CD5X04391IL2RGD11086PLOD2U84573
cdk inhibitor p27KIP1U10909IL6X04602PML-1M79462
C-foxNM_005252IL-8M28130PPP3CAL14778
CG12-1AF070675IL8RBL19593Pro. 4-hydroxyl.M24486
C-junJ04111iNOSL09210Prot.-ATPase-like pr.D89052
C-mycL00058Int-6U62962PTENU96180
C-mycV00568Integrin β 7M62880pyridoxal kinaseU89606
Collagen α1 (I)Z74615integrin-β-6NM_000888raf (c-raf-1)X03484
Collagen α2 (I)J03464IP-10X02530RAP46/Bag-1Z35491
Collagen, type XVI, alpha 1M92642IP-30J03909RbAp48X74262
Complement compound C1rJ04080IRF 1X14454ReticulocalbinD42073
COX17L77701IRF 4U52682RGS2NM_002923
Cpp32NM_004346IRF 5U51127RHONM_000539
CREBNM_004379IRF-1L05072RHO GDP-dis.inh. 2L20688
CTRL-1X71877IRF-2X15949RING 10NM_004159
CXCR4AF005058Irf-7U73036RING4X57522
Cyclin D1M64349ISG15AA406020Smad1U59423
Cyp19 (aromata)M28420ISG15M13755Smad2AF027964
Cys-X-Cys,member 11AF030514ISG-56KM24594Smad4U44378
DEAD box binding protein 1AF077951KIAA0129D50919Smad5U73825
DEAD-box protein p72U59321KIAA0235D87078Smad7AF015261
DestrinS65738KIAA0284AB006622SnoNX15219
DP (β1)M83664LIPAU04285SOCS 3/ssi-3AB004904
DR-αJ00194LMP-2X66401SOCS 4/CIS 4AB006968
E2F-1U47677L-selectinM25280SOCS1N91935
egr-1X52541Mad 4X03541SOCS-1NM_003745
Elastase 2M34379MAP2K1NM_002755SOCS2AF020590
ERMX76184MAP2K1IP1NM_021970SOCS-3NM_003955
F-actin capping proteinU56637MAP2K2L11285StanninNM_003498
Farn. pyro. syn.J05262MAP2K3NM_002756STAT 6U16031
FAS/Apo-1M67454MAP2K4L36870STAT1 (91kDa)M97935
fas-ligandU08137MAP2K5NM_002757STAT1 (91kDa)M97935
Fibronectin-1X02761MAP2K6U39657STAT2M97934
FK506 binding protein 6AF038847MAP2K7AF022805STAT4L78440
FKHRL1AF041336MAP3K1AF042838STAT5AL41142
Folate receptorX62753MAP3K11NM_002419STAT5BU47686
gadd45M60974MAP3K14NM_003954Succinyl CoA LigaseAF058953
Galectin-1J04456MAP3K2NM_006609TAP1 (Ring4)L21204
Gamma actinX04098MAP3K3U78876TFE3X96717
Gamma2-adaptin (G2AD)AF068706MAP3K4NM_005922TGF-bR1L11695
GAPDHX01677MAP3K5NM_005923TGF-bR2D50683
GATA 3X58072MAP3K7NM_003188TGF-bR3L07594
GBP-1M55542MAP4K1NM_007181TGIFX89750
GBP-2M55543MAP4K3NM_003618TIMP-1M59906
Granzyme BM17016MAPK10NM_002753TIMP-2J05593
GSK3NM_002093MAPK11NM_002751TIMP-3U14394
HCV-ass. p44D28915MAPK12NM_002969TIMP-4U76456
HLA-A (MHCI Ag B27)NM_002116MAPK13AF004709TNF-alphaX01394
HLA-EX56841MAPK14NM_001315TRAF6U78798
Homo sapins STATM97936MAPK3X60188TransferrinM12530
HouU32849MAPK6NM_002748TransthyretinD00096
HPAST proteinAF001434MAPK7NM_002749TRIP14L40387
hsf1 (tcf5)M64673MAPK8NM_002750trk oncogeneX03541
hsp90 (CDw52)X15183MAPK8IP2NM_012324TTF-2AF073771
Hypoxia-ind. Factor-1U22431MAPK9U35003UBE2L6AA292074
ICAM-1M24283MAPKAPK2NM_004759VCAM -1M30257
ICSB 1M91196MAPKAPK3NM_004635VEGF-CU43142
IDOM34455MCP-1/CCL2X14768Virpirin (Cig5)AF026941

In the established hepatoma cell line, hepG2.2.15 with stably transfected HBV genomes[16], ISG expression was examined using the cDNA macroarrays (Table 2). While many ISGs, e.g., 2-5 OAS, IFI 17, and RING4, were normally stimulated by IFN-α, several other ISGs were expressed at a lower level compared with the ISG expression in HepG2 cells. The induction of 2 ISGs, MxA and Cig5, was completely inhibited in HepG2.2.15 cells, while a partial inhibition was observed for 4 ISGs, IFITM1, IFITM2, IFITM3, and 6-16 (Table 1, Figure 1). Thus, only a subgroup of ISGs was down regulated in HepG2.2.15.

Figure 1
Figure 1 Northern blot analysis of ISG expression and its modulation by lamivudine in HepG2 and HepG2. 2.15 cells. HepG2 and HepG2.2.15 cells were cultured in the absence or presence of 2 μmol/L of lamivudine for 10 d. Then, the cells were stimulated with 100 or 1000 IU/mL of IFN-α for 6 h. Total cellular RNAs were isolated for Northern blotting hybridization. Lam: lamivudine.
Table 2 Suppression of ISG induction in HepG2.2.15 cells, effect of lamivudine treatment.
GeneAcc. No.HepG22.2.15HepG2/lam2.2.15/lam
I complete inhibition
MxAM338825.90.64.00.8
cig5AF0269412.20.92.11.2
II partial inhibition
IFITM3X573523.41.63.41.8
IFITM2X573512.71.62.21.8
III reversible inhibition
IFI 6-16BC0156037.94.47.26.5
IFITM1M245944.92.54.84.6
IV no inhibition
2-5OASD000683.84.04.45.8
MxBM308181.42.01.41.9
Caspase 7U673192.32.42.22.1
IFI 17J041643.32.92.82.7
IFI 27X673251.92.11.82.2
IFI T4U728822.71.92.61.8
RING4X575222.42.32.53.5
Analysis of the IFN response in HepG2.2.15 and HepG2 cells

The results above suggested that the IFN-signalling pathway is only partially inhibited in HepG2.2.15. Western blotting and EMSA and were carried out to analyse Stat1 activation and ISGF3 formation in HepG2 and HepG2.2.15 cells. The phosphorylated form of Stat1 was detected by western blot in IFN-α treated cells (Figure 2). The phosphorylation of Stat1 was enhanced in HepG2.2.15, compared with HepG2. Furthermore, Figure 3 showed that the formation of ISGF3 in HepG2.2.15 cells occurred after IFN-α stimulation, as occured in HepG2 cells. These data clearly show that the IFN-signalling pathway is generally not blocked in HepG2.2.15 cells. The results consistently show that both steps were evenly enhanced in HepG2.2.15. In addition, activation of ERK and p38 MAPKinase was not altered in HepG2.2.15 cells (data not shown).

Figure 2
Figure 2 Analysis of Stat1 phosphorylation after IFN-α stimulation in HepG2 and HepG2. 2.15 cells. Cells were stimulated with 100 U/mL of IFN-α for the indicated time points. Then, nuclear proteins were extracted and analysed by western blot. Data were quantified using Imagequant and are shown as relative units.
Figure 3
Figure 3 Analysis of ISGF3 formation after IFN-α stimulation in HepG2 and HepG2. 2.15 cells. HepG2 and HepG2.2.15 cells were stimulated with 1000 IU/mL of IFN-α for 6 h followed by isolation of nuclear extracts (NE-PERTM reagent kits) for EMSA analysis.
Reduction of the production of HBV proteins and the HBV replication by lamivudine treatment

The difference in ISG expression in HepG2 and HepG2.2.15 cell lines may be partly due to the presence of HBV replication in the later one. Consequently, the ISG expression in HepG2.2.15 would change if the HBV gene expression or replication is suppressed. To test this hypothesis, we determined the optimal condition to reduce HBV gene expression and replication using the nucleoside analogue lamivudine. HepG2.2.15 cells were treated with lamivudine at various concentrations from 0.04 μmol/L to 100 μmol/L. The antiviral activity was determined by quantitation of secreted HBsAg and HBeAg particles, extracellular virions and intracellular HBV replicative intermediates (RI). Figure 4A shows that treatment with lamivudine led to a significant reduction of secreted HBsAg and HBeAg in the supernatant of HepG2.2.15 cells. Parallel to the reduction of HBsAg and HBeAg production, the extracellular virion DNA in the culture supernatant of HepG2.2.15 cells and intracellular HBV replicative intermediates (RI) decreased after treatment with 2 μmol/L or 20 μmol/L of lamivudine for 10 d (Figure 4B and C). Maximal levels of suppression of HBV were observed after 10 d of lamivudine treatment. At that time, levels of RI were not more than 1.5% of controls in cultures of the 2 μmol/L treatment group. Based on these results, we chose a concentration of 2 μmol/L and duration of 10 d to suppress HBV replication in our system to study the modulatory effects of lamivudine on the IFN-response.

Figure 4
Figure 4 Antiviral effects of lamivudine in HepG2. 2.15 cells. A: HepG2.2.15 cells were cultivated with various concentrations (0 to 100 μmol/L) of 3 TC for 10 d. Then, supernatants were harvested and assayed for the presence of HBsAg and HBeAg by ELISA; B: HepG2.2.15 cells were treated with 2 or 20 μmol/L of lamivudine for 4, 7 and 10 d, respectively. Then, supernatants were collected and extracellular HBV-DNA was analyzed by dot blot hybridization; C: HepG2.2.15 cells were treated with various concentrations of lamivudine for 10 d. Then, intracellular HBV replicative intermediates were isolated for southern blotting. Lane 1: control, lane 2: 0.04 μmol/L, lane 3: 0.2 μmol/L, lane 4: 5 μmol/L, lane 5: 25 μmol/L, lane 6: 125 μmol/L, RC, relaxed circular HBV-DNA; DS, double stranded linear HBV-DNA.
The IFN response in HepG2.2.15 and HepG2 cells after lamivudine treatment

The effect of lamivudine on ISG expression in HepG2.2.15 and HepG2 was investigated by using gene macroarrays. No effect was observed for the stimulation of MxA and Cig5 expression by lamivudine treatment (Table 2). Both genes did not respond with an increased expression upon IFN-α stimulation. An increase of the IFN-α concentration to 1000 units per mL or a prolonged incubation with IFN-α did not change the expression of MxA and cig5. The reduced induction of IFITM 2 and IFITM 3 expression could not be enhanced by lamivudine treatment. In contrast, IFITM1 and 6-16 expression could be restored by lamivudine treatment of HepG2.2.15 cells (Table 1, Figure 1). This indicates that lamivudine can only partially normalize the IFN-response in HBV-transfected HepG2.2.15 cells at concentrations that profoundly inhibit viral replication and secretion of viral particles. Lamivudine had no effect on ISG expression in HepG2 cells and did not enhance the induction of many other ISGs, such as 2.5 OAS and MxB.

DISCUSSION

In the present work, we found that HepG2.2.15 and HepG2 respond differently to IFN-α. Several ISGs were not induced in HepG2.2.15 while they were expressed in HepG2 cells after IFN-α. There may be multiple reasons for the different ISG expression profiles in these cell lines, though HepG2.2.15 was derived from HepG2[16]. Previous data indicated that the expression of the IFN-inducible gene MxA was specifically inhibited by HBV proteins in HBV-transfected HepG2 or HuH7 cells[13], and this was accompanied by diminished antiviral activity of IFN[17]. In our study, we confirmed this finding with MxA expression being completely diminished in HBV-transfected HepG2.2.15 cells. In addition, we showed that additional ISG (Cig5, IFITM1, -2, -3 and 6-16) expression was completely abolished or partially reduced by HBV. The majority of ISGs, however, are expressed and inducible in both HepG2 and HepG2.2.15 cells, indicating that the HBV gene expression and replication had no effect on these ISGs. Consistently, Rosmorduc et al[17] demonstrated that 2´5OAS expression is not affected by HBV. Our results support the view that the HBV-mediated inhibition of the IFN-response, if any, represents a specific rather than global effect. The Stat1 activation or ISGF3 formation in HepG2.2.15 cells appeared to be normal, indicating that the Jak/Stat signalling pathway is intact and functional. These findings are corroborated by the data from Fernandez et al[13] who demonstrated that the inhibition of MxA induction in HepG2 cells occurs at the promoter level.

We then asked the question whether the HBV-mediated suppressive effect on the IFN-response could be reverted by treatment with the nucleoside analogue lamivudine, which is an effective inhibitor of HBV replication in vitro[18] and in vivo[19,20]. Lamivudine is phosphorylated within the cell and then incorporated into nascent viral DNA by the HBV polymerase during replication[21] resulting in the termination of HBV DNA elongation. Lamivudine also inhibits reverse transcriptase activity directly through competitive inhibition. Although some reports indicate that lamivudine exerts synergistic effects with IFN, the underlying mechanisms are not clear[22,23]. To answer this question we first established the optimal conditions for in vitro treatment of HepG2.2.15 cells with lamivudine. The results indicated that lamivudine exerted potent antiviral activities in our system as it strongly suppressed the formation of HBV replicative intermediates and extracellular HBV DNA at concentrations that correspond well to plasma levels found in patients that are treated with this drug. However, HBsAg and HBeAg secretion was only down regulated and not completely blocked. After treatment with lamivudine for 10 d, the induction of IFITM1 and 6-16 expression could be enhanced while MxA, Cig5, IFITM2 and IFITM3 induction remained unchanged. This indicates that lamivudine can at least partially improve the impaired IFN response in HBV-transfected cells. IFITM 1 to 3 and 6-16 belong to a family called small ISGs[24]. IFITM 1 to 3 are classified as members of the 1-8 group while 6-16 is a member of the ISG12 group. These genes were under the control of multiple elements responding to IFN-α stimulation including ISGF3 and interferon. It is likely that the lamivudine treatment partially reduces HBV gene expression and therefore contributes to the improved ISG expression. On the other hand, the continuing HBV protein production may still dominantly interfere with the expression of many ISGs, such as cig5 and IFITM3.

These findings are corroborated by our study that shows an improved IFN response of PBMC from HBV patients after treatment with adefovir. Some reports have also suggested a restoration of weak T helper cell and CTL responses after initiation of lamivudine therapy[25,26]. Although it is certainly a possibility, it still remains to be determined whether this effect can be explained by an enhanced responsiveness to IFNs.

In conclusion, our results suggest that HBV specifically modulates the IFN response in HepG2 cells by a selective suppression of certain ISGs. This suppression is at least partially reversible by antiviral treatment with the nucleoside analogue lamivudine.

Footnotes

S- Editor Wang J L- Editor Lutze M E- Editor Bai SH

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